Antibiotic BM408{60

ABSTRACT

This disclosure describes a new antibiotic, designated BM408 Alpha , produced in a microbiological fermentation under controlled conditions using a new strain of the microorganism Streptomyces canus.

United States Patent [191 Kirby et a].

[ ANTIBIOTIC BM408a [75] Inventors: Jane Parsons Kirby, New City;

Donald Bruce Borders, Suffern; Jean Hayes Korshalla, Pearl River, all ofNY.

[73] Assignee: American Cyanamid Company,

Stamford, Conn.

22 Filed: Feb. 25, 1974 21 Appl. No.: 445,176

[52] U.S. Cl. 260/210 AB; 195/80; 424/181 [51] Int. Cl. C07H 15/22 [58]Field of Search 260/210 AB, 210 R, 210 K, 260/210 NE Dec. 23, 1975Primary Examiner-Johnnie R. Brown Attorney, Agent, or FirmEdward A.Conroy, Jr.

57 ABSTRACT This disclosure describes a new antibiotic, designatedBM408a, produced in a microbiological fermentation under controlledconditions using a new strain of the microorganism Streptomyces canus.

5 Claims, N0 Drawings ANTIBIOTIC BM408a BRIEF SUMMARY OF THE INVENTIONCH OH HO NH HO NHZ OH Ch OH HO OH The present invention includes withinits scope the antibiotic in dilute form, as a crude concentrate, and inpure crystalline form. The structure of this new antibiotic as well asits effects on specific microorganisms differentiate it from previouslydescribed antibacterial agents.

The novel antibiotic of the present invention forms acid-addition saltswith a variety of pharmaceutically acceptable organic and inorganicsalt-forming reagents. Thus, acid-addition salts, formed by admixture ofthe antibiotic base with one, two, or three equivalents of an acid,suitably in a neutral solvent, are formed with such acids as sulfuric,phosphoric, hydrochloric, hydrobromic, nitric, citric, lactic, tartaric,acetic, and related acids. For purposes of this invention, theantibiotic free base is equivalent to its nontoxic acid-addition salts.

DETAILED DESCRIPTION OF THE INVENTION This new antibiotic BM408a isformed during the cultivation under controlled conditions of a newstrain of the microorganism Streptomyces canus. This new antibioticproducing strain was isolated from a range soil sample collected nearBoise, Id. A viable culture of the new microorganism has been depositedwith the Culture Collection Laboratory, Northern Utilization Researchand Development Division, United States Department of Agriculture,Peoria, III., and has been added to its permanent collection. It isfreely available to the public in this depository under its accesionnumber NRRL 5781.

The description and identification of this new microorganism, maintainedin the culture collection of the Lederle Laboratories Division, AmericanCyanamid Company, Pearl River, NY. as Culture No. BM408, was supplied byDr. l-l.D. Tresner of these laboratories. The following is a generaldescription of the microorganism Streptomyces canus NRRL 5781, based ondiagnostic characteristics observed. Observations were made of thecultural, physiological and morphological features of the organism inaccordance with the methods detailed by Shirling, E. B. and Gottlieb,D., Methods for Characterization of Streptomyces Species, InternationalJournal of Systematic Bacteriology, 16: 3l3-340 (1966). The underscoreddescriptive colors and color chip designations were taken from Jacobson,E., et al., Color Harmony Manual, 3rd edition (1948), ContainerCorporation of America, Chicago, Illinois.

Amount of Growth Moderate to heavy on all media used except Bennetts andCzapeks Solution Agars on which only light growth developed.

Aerial Mycelium and Sporulation Aerial mycelium whitish, becoming SilverGray (3 fe) to Ashes (5 fe) in sporulating zones on most media.

Soluble Pigments Yellowish to yellowish-brown 0r brownish on most mediaand in light to moderate amounts; absent on Benedicts, Bennets andInorganic Salts-Starch Agars.

Reverse Color Yellowish-brown to brownish shades.

Miscellaneous Physiological Reactions Complete liquefaction of gelatinin 14 days; nitrates partially reduced to nitrites in 7 days; completepeptonization of purple milk, no curd formation; melaniod pigments notformed on peptone-iron agar; NaCl tolerance in yeast extract agar 7% but10%. Carbon source utilization, according to the method of Pridham, T.G. and Gottlieb, D., The Utilization of Carbon Compounds by SomeActinomycetales as an Aid for Species Determination, Journal ofBacteriology 56: 107-1 14 (l948) as follows: Good utilization ofglycerol, i-inositol, lactose, d-mannitol, d-melibiose, d-raffinose,l-rhamnose, salicin, sucrose, d-trehalose, d-xylose and dextrose; fairutilization of d-fructose; poor utilization of adonitol, l-arabinose andd-melezitose.

Micromorphology Aerial mycelium gives rise to spore chains in clustersand tangles of tightly wound coils and spirals of a few to many turns.Spores mostly globose to elliptical; 1.0-1 .2p.m 0.6O.8p.m; sporesurfaces spiny as determined by transmission electron microscopy; spinesslender, up to 0.5um in length.

Diagnosis Culture BM408 is a member of the gray-spored streptomyceteswhich have, in common, spiralled spore chains, spiny spores and a lackof melanoid pigments. There are approximately two dozen validlydescribed species in this group. Culture BM408 has been compared withpublished descriptions of these species and, whenever possible, withactual reference specimens. After careful consideration of all membersin the group, it was determined that BM408 most closely corresponded tothe species Streptomyces canus. In the absence of any outstandingdiagnostic differences, BM408 will therefore be considered a strain ofthat species.

It is to be understood that for the production of antibiotic BM408oz,the present invention is not limited to this particular microorganism orto microorganisms fully answering the growth and microscopiccharacteristics which are given for illustrative purposes. In fact, itis desired and intended to include the use of mutants produced from thedescribed microorganism by various means such as exposure toX-radiation, ultraviolet radiation, nitrogen mustard, actinophages, andthe like.

The novel antibiotic of the present invention as well as several relatedantibiotics may be represented by the following general formula:

CH R NH F0 Nu, OH

cu OH R2 2 wherein BM408a is represented when R, OH; R H; R OH; and R H.Thus BM4-08a is a structurally related to vistamycin (also calledAntibiotic SF733 or ribostamycin), E. Akita, T. Tsuruoka, N. Ezaki andT. Niida, Journal of Antibiotics, 23, 173 (1970), wherein R NH R H; ROH; and R H; an antibiotic currently in clinical study in Japan; and toBu1709l3 (wherein R OH; R OH; R H; and R COCH- (OH)CH CH NH and BU l709E (where R OH; R H; R OH; and R COCH(OH)CH CH NH H. Tsukiura, K.Saito, S. Kobaru, N. Konishi and H. Kawaguchi, Journal of Antibiotics26, 386 (1973).

The fermentation of Streptomyces canus NRRL 5781 also produces BM408Band a minor component BM408'y which were identified as known antibioticsparomomycin l and paromomycin 11, respectively.

Ell/14080. is a white, water soluble powder having [oz],,""= +30(i 2or(C 0.0995, H O). The mass spectral data for Eli/1408a are presented inTable III.

Fermentation Process Cultivation of Streptomyces canus NRRL 5781 may becarried out in a wide variety of liquid culture media. Media which areuseful for the production of the novel antibiotic BM408a include anassimilable source of carbon such as starch, sugar, molasses, glycerol,etc.; an assimilable source of nitrogen such as protein, proteinhydrolysate, polypeptides, amino acids, corn steep liquor, etc.; andinorganic anions and cations, such as potassium, sodium, calcium,sulphate, phosphate, chlo ride, etc. Trace elements such as boron,molybdenum, copper, etc. are supplied as impurities of otherconstituents of the media. Aeration in tanks and bottles is provided byforcing sterile air through or onto the sur- 4 face of the fermentingmedium. Further agitation in tanks is provided by a mechanical impeller.An antifoaming agent, such as lard oil, may be added as needed.

Inoculum Preparation inoculum of Streptomyces canus NRRL 5781 may beprepared by growing the organism in a medium of the followingcomposition:

Corn starch 24 gm./liter Bacto tryptone 5 gm./liter Yeast extract 5gm./liter Beef extract 3 gm./liter Dextrose l gmjliter Tank FermentationA 3 to 10% portion of the above inoculum is used to seed 300 liters ofmedium of the following formulation:

Dextrose l5 gm./liter Glycerol l5 gm./liter Soy flour 15 gm./literCalcium carbonate 1 gm./liter Sodium chloride 3 gm./liter NaOHSufficient to adjust pH to 7.0

Aeration is supplied at the rate of 0.2 to 0.8 liter of sterile air perliter of broth per minute and the mash is agitated by an impeller drivenat 200 to 400 r.p.m. The temperature is maintained at 32 to 40C.,usually at 37C. The fermentation is ordinarily continued for to 120hours, at which time the mash is harvested.

Isolation and Purification of Antibiotic BM408a After the fermentationis completed, the fermented mash containing BM40801 is filtered. Thefiltrate is passed through a column of Amberlite XAD-Z (a polystyrenecross-linked with divinyl benzene) resin. The column effluent is thenpassed through a column of Amberlite RC-5O (a methacrylic acid-divinylbenzene ion exchange resin) (NHX) resin. The resin is washed with waterand the antibiotic is eluted with 2N NH OH. The column eluate (pH 11.7)is evaporated under reduced pressure to about ml. and placed on a columnof Dowex 1-X2 (a trimethylbenzylam'monium polystyrene cross-linked with2% divinyl benzene) (Oi-l") resin which is eluted with water. Theantibiotic activity is detected by in vitro assay of a 0.1 ml aliquot ofeach fraction against Klebsiella pneumoniae in the conventional agardiffusion assay. The antibiotic activity is detected in the 255 to 935ml. fractions of effluent. These fractions are pooled and concentratedon a rotary evaporator to a thick, yellow viscous liquid. The antibioticis adsorbed onto Dowex 50 W-XS (a sulfonated polystyrene cross-linkedwith 8% divinyl benzene) (l-l) resin. The column is rinsed with waterand 7 Streptococcus pyogenes (NY i Staphylococcus aureus (ATCC 14154) 5the activity is eluted with 1.5 N NH OI-I in 475-814 ml. of eluate. Aconcentrate of the ammonium hydroxide eluates is chromatographed on aDow ex'1-X2 (OI-l) column. The column iselutedwith watriyielding threemajor fractions. Fraction I is adsorbed'on a column of Amberlite CG-50(a methacrylic acid-divinyl benzene ion exchange resin) (Nl-l The resinis eluted with water, 0.1N NH OH and finally'OQBN INI-I OI-I using astepwise developmentTwo bioact'iveeluates are obtained, BM408a (170-220ml.) and a mixture of BM408B and 7 (265-3 15 ml.). The BM408a eluate istaken to asrnall volume on a rotary evaporator, freeze dried and theproduct, BM408a, is recovered as a fine white powder. I

In vitro Activity Antibiotic BM408a is active in vitro against a varietyof gram-positive and gram-negative bacteria when tested by thestandar'dagar well diffusion assay procedure. The results of such a testappear in Table I and are reported in terms of the zone of inhibition.

Table 1 Zone of Bacillus cereus (Waksman) Klebsiella pneumoniae(Friedlanders) Alcaligenes sp (ATCC 1015 3) Bacillus subtilis (StanslyR-78) Bacillus subtilis (pH 6.0 agar) Bacillus .rubtilis (Stansly R-76)Mycobacter ium smegmatis (Stansly R-99) Mycobacte rium smegmatis (pl-I6.0) Staphylococcus aureus (No. 208) Escherichia coli (Parke, Davis)Escherichia coli (resistant to Chloi amphenicol) Staphylococcus aureus(209 1) j Carynebaclerium xerosis (NRRL 8-1397) Salmonella gallinarum(No.1605) Salmonella gallinarum (pl-I 6.0) Staphylococcus aureus (Smith)Klebsiella pneumoniae (AD) Candida albicans (CA 300) Pseudamonasaeruginosa (ATCC 10145) Escherichia. coli (31 I Aerobacter aerogenes(75) Proteus mirabilis Escherichia coli (311 DY) Cryptacoocus neafarmans(SP) Distance from edge of well to outer edge of inhibition zone In vivoActivity The antibiotic BM408a is active in vivo against Proteusmirabilis. This new antibacterial is thereby potentially useful as atherapeutic agent in treating bacterial infections in mammals. This newantibacterial'can be expected to be usefully employed for treating orcontrolling bacteria infections by parenteral administra tion. Theusefulness of this new antibacterial agent is demonstrated by itsability to control systemic lethal infections of Proteus mirabilis 4671in mice when administered by "a single 0.5 ml. subcutaneous dosesuspended in 0.2% aqueous agar to groups of Carworth Farms CF-l femalemice weighing 18-22 gms., which have been infected intraperitoneallywith a lethal dose consisting of 0.5 ml. of a 10 tryptlcase soy brothdilution of a 5 hours trypticase soy broth culture of Proteus mirabilis4671. Table 11 illustrates the results.

6 Tablell Alive/Total Mice Tested Single Subcutaneous 7 Days AfterInjection Dose (mg/kg.)

64 1/2 32 0/2 Infected non-treated controls I/ 20 The invention will bedescribed in detail in conjunction with the following specific examples.

Corn starch 24 gm./liter Bacto tryptone 5 gm./liter Yeast extract 5gm./liter Beef extract 3grn./1iter Dextrose 1 gmJIiter pH adjusted to7.0 with NaOH The washed or scraped spores from an agar'slant of cultureStreptomyces canus NRRL 5781 were used to inoculate each of two 500 ml.flasks each containing 100 ml. of the above sterile medium, which hadbeen adjusted to pH 7.0. The flasks were placed on a rotary shaker andagitated vigorously for 48 hours at 28C.

The resulting flask inoculum was transfered to a 5 gallon glass.fermentor containing 12 liters of the same sterile medium. The inoculummash was aerated with sterile air while growth was carried out for about48 hours at 28" C., after which the contents were used to seed a 300liter tank fermentor.

EXAMPLE 2 Fermentation A fermentation medium was prepared according tothe following formula:

Dextrose Glycerol 15 gm./liter Soy flour l5 gm./liter Calcium carbonate1 gmJliter Sodium chloride 3 gmJliter The pH is adjusted to 7.0 withNaOH EXAMPLE 3 Isolation and Purification of BM408a The 300 literportion of harvested mash from Example 2 was filtered and the resultingfiltrate, at pH 8.0, was passed through a 3 liter bed volume or 10.16cm.

by 37 cm. column of Amberlite XAD-2 resin. The

column effluent was then passed through a 5 liter bed volume or 10.16cm. by 61.7 cm. column of Amberlite [RC-50 (NHJ) resin. A monitor forantibiotic activity against Klebsiella pneumoniae by the agar diffusionassay was set at 4 hour intervals. The lRC-50 resin was washed with 15liters of water and the antibiotic was eluted with 30 liters of 2N Nl-lOl-l. The column eluate, consisting of 30 liters at pH 1 1.7, wasevaporated under reduced pressure to 100 ml. This 100 ml. was passedthrough a 3 X 52 cm. column of Dowex 1-X2 (OI-l) (50-100 mesh) resin.The column was eluted with water and the antibiotic activity wasdetected by in vitro assay of 0.1 ml., aliquots of each fraction againstKlebsiella pneumoniae. Antibiotic activity was detected in the 255 ml.to 935 ml. fractions of column effluent and these fractions were pooledand concentrated on a rotary evaporator to 22 ml. of thick, yellowviscous liquid. The antibiotic in the concentrate was adsorbed onto acolumn of Dowex 50 W-X8 (H resin, the column was washed with water andthe activity eluted with 1.5N Nl-l OH in 475-814 ml. of column eluate. A10 ml. concentrate of the ammonia eluate was chromatographed on a 1.5 X20 cm. Dowex 1-X2 (OH) (200-400 mesh) column. The column was eluted withwater and three major fractions (40-70, 80-90, and 100-210 ml. of columneffluent) were recovered, with solids totaling 868.3 mg.

A 550 mg. portion of the above material (first fraction) was adsorbed onan Amberlite CG-50 (NI-1 (100-200 mesh) (1.5 X 20 cm.) column. Thecolumn was eluted, in order, with 300 ml. portions of water, 0.1N NH OHand 0.3N NH OH using a stepwise development. Two bioactive eluates wereobtained, BM40801 at 170-220 ml. and a mixture of BM408B and 7 at265-315 ml. The BM408a eluate was reduced to a small volume on a rotaryevaporator, freeze-dried and the solid was recovered as a fine whitepowder. Yield 157.0 mg. [a],, +30 (i 2 or 10) (C 0.0995, H O) Table 111Relative Abundance and Composition of Selected Ions in the Mass Spectrumof the N-Acetyl- O-trimethylsilyl Derivative of BM408a Table[ll-continued Relative Abundance and Composition of Selected t fth tlb-i'r'im m lsii l ii ri iiv of BM40%1 Relative Observed CalculatedAbundance Mass Mass Composition 22 649.3177 649.3192 0 H, N Si 737.3284737.3352* cj h m fist. 57 767.3602 767.3642 2 ,0 ,5 33 9133312 382-231?22 1040:4664 1040:4670" ciiufiujolsgi 14 1070.4948 1070.4958C43H92N3O|4SI1 Ions resultin from inadvertant O-acet lation of thehydroxyl group of the deoxystreptamine moiety.

We claim: l. A compound selected from the group consisting of thecompound of the formula:

and the pharmacologically acceptable acid-additon salts thereof.

2.. The antibiotic free base according to claim I.

3. The antibiotic monohydrochloride according to claim 1.

4. The antibiotic dihydrochloride according to claim 5. The antibiotictrihydrochloride according to claim

1. A COMPOUND SELECTED FROM THE GROUP CONSISTING OF THE COMPOUND OF THEFORMULA:
 2. The antibiotic free base according to claim
 1. 3. Theantibiotic monohydrochloride according to claim
 1. 4. The antibioticdihydrochloride according to claim
 1. 5. The antibiotic trihydrochlorideaccording to claim 1.